Composite gel microparticles as active principle carriers

ABSTRACT

The invention relates to vectors for delivering medicinal, nutritional, plant-protection or cosmetic active principles, these delivery particles being of small, controllable and adjustable particle size, which protect the active principle, and being biocompatible, biodegradable, non-immunogenic, stable and free of solvent. The particles do not denature the active principle and allow the active principle to be released. The microparticles of the invention are of a cohesive structure made of a physicochemically stable and integral composite gel which includes an oil such as coconut oil, an aqueous phase and a linear, non-crosslinked copolyamino acid of Leu/Glu type (random or diblock). The microparticles have a controllable and adjustable size of between 0.05 and 500 μm.

This application is a 371 of PCT/FR97/00471 filled Mar. 14, 1997.

BACKGROUND OF THE INVENTION

The field of the present invention is that of vectors which are usefulfor the administration of active principles (APs), preferably medicinalor nutritional active principles, in particular via the oral, nasal,vaginal, ocular, subcutaneous, intravenous, intra-muscular, intradermal,intraperitoneal, intracerebral, parenteral, etc. route. These vectorsallow the protected delivery of the APs into the body, to their site ofaction. They are intended to improve the bioavailability of APs. Thesevectors can be, for example, systems for the sustained release of APs.

The APs with which the invention is more particularly, but notexclusively, concerned are, for example, proteins, glycoproteins,peptides, polysaccharides, lipopolysaccharides, oligonucleotides andpolynucleotides.

More specifically, the present invention relates to delivery particles(DP)—advantageously of submicron and/or micron type—for delivering APs,in particular medicinal APs.

Besides medicinal and nutritional APs, the invention also relates toplant-protection and cosmetic APs.

The present invention is directed toward both naked particles per se,and AP delivery systems, consisting of particles loaded with the AP(s)considered.

The invention also relates to a process for preparing the saidparticles.

PRIOR ART

Progress in genetic engineering and biotechnology, as well as therelated discoveries of biologically active peptide, proteinic andgenetic tools have allowed the development of novel medicinal activeprinciples (APs) offering high intrinsic activity and high selectivity.On the other hand, these APs are easily degraded in the body beforereaching their therapeutic site of action, and their bioavailability isconsequently very low. In the case of oral administration, thegastrointestinal tract constitutes a formidable chemical and physicalbarrier for an AP which must, on the one hand, withstand degradation bythe digestive system and, on the other hand, pass through thegastrointestinal epithelial membrane. In this respect, reference may bemade, for example, to the review by M. J. Humphrey (Delivery System forPeptide Drugs, edited by S. Davis and L. Illum, Plenum Press, N.Y.,1986), which gives an account of the low bioavailability of peptides andof peptides administered orally.

Naturally, these mishaps of delivery and residence in the body are notlimited to proteins, but also affect APs formed by genetic tools(oligonucleotides, polynucleotides, plasmids) liable to be used ingenetic engineering techniques.

To overcome this, it has been proposed to encapsulate APs in AP deliveryparticles, also referred to as DPs. The advantage of these encapsulationtechniques is that they protect and/or deliver the AP up to itstherapeutic site of action, by keeping it safe from attack by the body,in order to increase its bioavailability.

Of all the materials which can be envisaged for encapsulating APs,polymers are increasingly used on account of their intrinsic properties.

As regards the list of specifications which it is desired to obtain forsuch DPs, this is particularly demanding and comprises, in particular,the following specifications:

1—It should, advantageously, be possible to obtain DPs with an averagediameter of between a fraction of a micron and a few microns, with anarrow particle size distribution, so as to be able to adapt the DPparticle size to the mode of administration selected and/or thetherapeutic site targeted. For example, if a mucosal immunization viathe oral route is desired, the DP size should be between 0.5 μm and 10μm in order for the DPs to be able to penetrate the Peyer plates andreach the lymphoid tissues. In the case of subcutaneous administration,it is advantageous to have DPs larger than 10 μm in size in order forthe particles not to enter into the general circulation, in which theyare rapidly internalized by the reticuloendothelial system, but for themto diffuse gradually from their site of injection.

This specification implies controlling the size of the DPs, as regardsboth the DP particle size distribution and their average diameter, whichrepresents a very intricate operation in technological terms.

2—It is desirable for the DPs to protect the AP up to the site ofrelease. For example, in an oral administration of an AP formed of avaccine, the vaccine would be successfully protected throughout thegastrointestinal tract.

3—It is preferable for the polymer which forms the DPs to bebiocompatible and biodegradable and, better still, for it to bemetabolized into products that are non-toxic to the body.

4—It is also advantageous for the polymer which forms the DPs not toinduce an immune response (immunogenic).

5—The DPs are expected to allow the controlled and sustained release ofthe AP.

6—Lastly, it is also preferable for the DPs to be obtainable by aprocess which does not denature the AP. Thus, the use of organicsolvents and/or high temperatures should be ruled out.

Many prior technical approaches have attempted, unsuccessfully, tosatisfy all of these specifications. The solutions provided hitherto arethus only partial and incomplete.

In all of these unfruitful approaches of the prior art, severalconstituent base materials have been envisaged for the deliveryparticles. These materials can be biocompatible polymers, such asproteins and/or polymers of α-hydroxy acids (polylactic and/or glycolicacids) and/or poly(alkyl cyanoacrylates) and/or polyorthoesters and/orfatty substances (oils—fats).

The DPs (microspheres or microcapsules) of protean nature are usuallyobtained by drastic crosslinking treatments using glutaraldehyde-typechemical agents or by raising the temperature. It goes without sayingthat such treatments necessarily entail denaturing of a large number ofactive principles. What is more, the toxicity of glutaraldehyde-typecrosslinking agents is particularly unfitting in pharmaceuticalapplications. As examples of such known microparticles, mention may bemade of those disclosed in patent application EP 0,363,921. Theparticles according to that application are obtained by complexcoacervation of a synthetic polyamino acid and an anionic polymer, inaqueous solution, by adjusting the pH. The polyamino acid is anamphiphilic copolymer based on glutamic acid and lysine, whereas theanionic polymer is a water-soluble polysaccharide, such as gum arabic.These particles are given cohesion by crosslinking the coacervate usingglutaraldehyde. Besides the toxicity and degradation problems of the APsmentioned above, it should be noted that the polymers in the coacervateaccording to EP 0,363,921 suffer from an immunogenic nature.

Protean microparticles or microparticles based on biocompatible andbiodegradable polymers can also be prepared by standard emulsiontechniques.

In this respect, mention may be made, for example, of patentapplications WO 91/06 286 and WO 91/06 287 which describe processes forthe formation of particles in emulsion, these processes using, aspolymer:

either a hydrophobic protein chosen from collagen, casein, keratin and,preferably, prolamines,

or a biocompatible and biodegradable polymer, such as poly(lactics) orpoly(orthoesters).

The AP can be hydrophobic or hydrophilic but, in the latter case, thedouble-emulsion technique is recommended. The size of the microparticlesis about 100 μm and preferably between 50 nm and 100 μm.

Patent application WO 89/08 449 also makes reference to encapsulation byemulsion, in order to incorporate APs into poly(lactic) microparticlesless than 10 μm in size. Moreover, it is pointed out in that documentthat this size is a maximum limit for absorption across the lymphoidtissues of mucous membranes (oral, nasal, rectal and ophthalmologicaladministrations).

The emulsion techniques are very appealing in principle, since theyallow most of the APs to be used in microparticles whose particle sizecan be controlled to sizes of about 1 μm. However, in these techniques,organic solvents are used to dissolve the polymers which make up theparticles. These solvents are, for example, ketones, alcohols, amides ormixtures thereof. Unfortunately, in addition, it turns out that thesesolvents can be denaturing, in particular for peptide or polypeptideAPs.

Mention will also be made, with respect to the constituent polymers ofpoly-α-hydroxy acid type (polylactic and/or glycolic type) of a problemof accumulation in vivo which is liable to give rise to rejectioneffects.

Biocompatible DPs formed in aqueous solution without excessively raisingthe temperature, and referred to as proteinoids, are also known. TheseDPs were described as early as 1970 by W. Fox and K. Dose in “MolecularEvolution and the origin of Life”, Ed. Marcel Dekker Inc. (1977).

Taking inspiration from these studies, patent application Wo 88/01 213proposes a proteinoid-based AP delivery system. The polymer used is amixture of artificial polypeptides obtained by thermal condensation ofnatural or synthetic amino acids and/or of small peptide chains. Themode of condensation chosen leads to branched oligomers which are thusvery sparingly soluble. A selection is then made by filtering thesebranched oligomers, in order to recover the water-soluble fractions.This fraction is necessarily composed of branched reticulates of verylow mass. The microparticles according to that invention are obtained bychanging the pH which causes precipitation of the branched oligomers asproteinoids.

When the solution in which the precipitation is carried out contains APsin solution, some of them are entrained into the proteinoid when it isformed. The drawbacks of this system are:

a low degree of encapsulation,

a method of synthesis by lowering the pH,

an intricate process for purifying the polymers,

an irregular (non-alpha-peptide) amino acid sequence due to the mode ofsynthesis which makes it possible to assert only that the enzymaticdegradation reactions will be identical to those of an α-polyamino acid,

lastly, the use of a large number of different amino acid monomers,which can induce an immune response.

Patent application WO 93/25 589 relates to an improvement to the processfor the synthesis of proteinoids by thermal condensation of amino acids.The same drawbacks as those associated with the subject-matter of theparent application mentioned above are found.

The nanocapsule-type microparticles described in patent application EP0,608,207 and whose wall is based on poly(alkyl 2-cyanoacrylate), arementioned here merely as a matter of interest, since the toxicity of theresidues of the monomers used is clearly detrimental for the delivery ofAPs in man and/or animals. Moreover, little is known about theelimination of the residues of such polymers.

Now, as regards DPs of essentially lipidic or hydrophobic nature,several types of systems may be listed, namely:

liposomes,

solid lipid particles (SLPs),

multi-chamber lipid vesicles (MCLVs),

supercooled molten particles (SMPs),

lipid emulsions,

and particles of the lipid matrix carrier (LMC) type.

Liposomes are spherical colloidal structures comprising an aqueousinternal phase enveloped by one or more phospholipid bilayers. Liposomesare known as delivery particles.

A first drawback of liposomes is their instability in biological fluidsand the high rate of release of the AP which they may contain. Referencemay be made in this respect to the article by Kim et al., Biochim.Biophys. Acta, 728, 339-348, 1983.

A second drawback of liposomes is that they only allow a low level offilling with AP.

A third drawback of liposomes relates to their instability on storage.

A fourth drawback of liposomes is associated with the poorreproducibility of their manufacture and, in particular, their mediocreability to trap APs.

There is a wealth of patent literature regarding liposomes. Mention willbe made, as a matter of interest, of the following patents: U.S. Pat.Nos. 3,993,754, 4,235,871, 4,356,167 and 4,377,567.

International patent application PCT WO 94/20 072 describes solid lipidparticles (SLPs) of non-spherical form, consisting of a crystallinelipid matrix which is solid at ambient temperature. The high-meltinglipids concerned are preferably triglycerides (θ fusion=30-120° C.). Insuspension, these SLPs can be stabilized by amphiphilic, ionic ornonionic compounds. These amphiphilic stabilizing compounds can bephospholipids, sphingolipids, glycosphingolipids, physiological bilesalts, saturated or unsaturated fatty acids, fatty alcohols,methoxylated fatty acids or alcohols, as well as esters thereof andethers thereof, polyether alkyloaryl alcohols, esters and ethers ofsugars or of sugar-alcohols with fatty acids or fatty alcohols,acetylated or ethoxylated mono- or diglycerides, block copolymers ofpolyoxyethylene and of polyoxypropylene oxide, ethoxylated sorbitanethers or esters, amino acids, polypeptides, proteins (gelatins,albumins), as well as mixtures of the abovementioned compounds.

On account of their crystalline solid nature, these SLPs necessarilyinclude a step of melting at high temperature, during their formationand the incorporation of the AP. It has already been pointed out thatheating to high temperatures is harmful to certain sensitive APs. Inaddition, given the exclusively lipidic nature of SLPs, they turn out tobe unsuitable for hydrophilic APs. Lastly, when a hydrophobic APdifferent in nature from the one forming the matrix of the SLP isconcerned, the incorporation yields are extremely low, of the order of afew %.

International patent application PCT WO 95/13 796 describes internalaqueous multi-chamber lipid vesicles (MCLVs). These DPs are novel withrespect to liposomes by virtue of their non-concentric multi-chamberstructure and their aqueous content. The said aqueous chambers are eachformed of a membrane formed of a lipid bilayer which defines a sphere.

In order to prepare these MCLVs, a neutral lipid of plant oil, animalfat or tocopherol type is used, as well as an amphiphilic lipid whichhas a large negative charge, e.g. phosphatidylserine. These two types oflipids are dissolved in an organic solvent to which is added an aqueoussolution comprising the AP to be encapsulated, so as thus to form awater-in-oil emulsion. This emulsion is completed by adding an agent fordelaying the release of the AP. A second emulsification is then carriedout by adding a second aqueous solution containing at least one nonionicosmotic agent and an acid-neutralizing agent of low ionic strength.After stirring, spherules of solvent containing many aqueous dropletsare obtained. On evaporating the solvent, these spherules are convertedinto MCLVs. The inner aqueous chambers are thus in suspension in thesecondary aqueous solution and not in the chloroform which has beeneliminated.

Like its alternative forms, this double-emulsion technique can becriticized in that it involves toxic organic solvents whose absence, intrace form, from the final microparticles cannot be guaranteed.

International patent application PCT WO 95/05 164 describes particles ofubidecarenone or of other substances which have poor water solubility,in which this or these substances with poor water solubility are in the“supercooled molten” state, i.e. they are in a state similar to theliquid state at a temperature below their melting point. Theseparticles, also known as SMPs, require the use of an amphiphilicstabilizer (lecithin) when they are suspended in an aqueous liquid.These stabilizers are the same as those described in patent applicationPCT WO 94/20 072 which concerns SLPs. The supercooled molten substanceforming the particle has a melting point of about 70° C. These are thusfats rather than oils that are liquid at ambient temperature. The fattysubstances capable of forming such a molten substance are chosen fromvitamins, sterols and triglycerides, for example.

These SMPs are obtained by melting the substance intended to form thesupercooled molten substance. The amphiphilic compound is then dispersedin this molten substance. Water is then added and the mixture issubjected to homogenization/stirring at high speed, while at the sametime keeping the reaction temperature above the melting point of thesubstance used. A dispersion of SMP in a continuous aqueous phase isfinally obtained. The SMP microparticles are free, or virtually free, ofwater.

Such vectors have the drawback of being specific and exclusive tohydrophobic APs. In addition, the high temperatures required to carryout the process for preparing these SMPs can be harmful to the APs.

Delivery systems involving lipid emulsions consisting of droplets ofliquid oil dispersed in an aqueous phase and stabilized by aninterfacial film of emulsifier (lecithins) are also known. AP deliveryusing DPs, constituting the heterogeneous phase of the lipid emulsion,is described in international patent application PCT WO 91/02 517. Thecohesion, integrity or stability of such DPs is precarious or fragile,since these DPs have no physical existence, i.e. they do not formdefinite substances outside of the emulsion medium. Once the equilibriumestablished by the surfactant is disturbed, the droplets of liquid oilcoalesce and disappear. Consequently, these lipid emulsions offer veryfew concrete measures in AP delivery.

U.S. Pat. No. 4,610,868 relates to a novel type of lipid DP, which canbe described as being a globular structure formed of a lipid matrix.These DPs, also known as LMCs, are between 0.5 μm and 100 μm in size.The base constituents of the LMCs are a hydrophobic compound, anamphiphilic compound and, optionally, an AP accompanied by water if thelatter is of water-soluble nature.

The hydrophobic compound is preferably a triglyceride-based oil, such ascorn oil or coconut oil. Sterols may also be suitable as hydrophobiccompounds.

The amphiphilic compounds recommended are those of lipidic nature, suchas phosphoglycerides and, more particularly, phosphatidylcholine fromegg yolk.

The preparation of these LMCs involves an organic solvent, such asacetone or ethanol. According to a specific embodiment of the processfor preparing these LMCs, a water-in-oil emulsion—obtained fromphosphatidylcholine oil, water and water-soluble APs—is extruded intothe organic solvent with stirring. The LMCs are generated at the momentthat the W/O emulsion is introduced into the solvent. When the AP ishydrophobic, the mixing of amphiphilic oil and of AP takes place in theorganic solvent, the said mixture then being extruded into an aqueousphase.

For water-soluble APs, it can be envisaged to totally eliminate theaqueous phase and to extrude the oil+amphiphile+AP mixture into theorganic solvent. After elimination of the organic or aqueous solvent,LMCs are recovered which, according to the patent, are described asresembling droplets of oil which do not coalesce.

These LMCs have the major drawback of requiring the use of solventsduring their preparation, and it is known that the presence of solvent,even in trace amounts in DPs, is particularly undesirable. Theexclusively lipidic nature of LMCs will be stressed. Lastly, it shouldbe considered that the process for obtaining the LMCs is relativelycomplex and hardly convenient to carry out industrially (extrusion intoa solvent).

SUMMARY OF THE INVENTION

In this state of understanding, one of the essential aims of the presentinvention is to provide DPs, in particular submicron-sized andmicron-sized DPs, based on lipids and which can serve as vectors for anactive principle (AP), in particular a medicinal and/or nutritional AP,in order to administer the said AP to a human or animal body, these DPsfully satisfying the list of specifications given above and repeatedbelow:

1—It should, advantageously, be possible to obtain DPs with an averagediameter of between a fraction of a micron and a few microns, with anarrow particle size distribution, so as to be able to adapt the DPparticle size to the mode of administration selected and/or thetherapeutic site targeted. For example, if a mucosal immunization viathe oral route is desired, the DP size should be between 0.5 μm and 10μm in order for the DPs to be able to penetrate the Peyer plates andreach the lymphoid tissues. In the case of subcutaneous administration,it is advantageous to have DPs larger than 10 μm in size in order forthe particles not to enter into the general circulation, in which theyare rapidly internalized by the reticuloendothelial system, but for themto diffuse gradually from their site of injection.

This specification implies controlling the size of the DPs, as regardsboth the DP particle size distribution and their average diameter, whichrepresents a very intricate operation in technological terms.

2—It is desirable for the DPs to protect the AP up to the site ofrelease. For example, in an oral administration of an AP formed of avaccine, the vaccine would be successfully protected throughout thegastrointestinal tract.

3—It is preferable for the polymer which forms the DPs to bebiocompatible and biodegradable and, better still, for it to bemetabolized into products that are non-toxic to the body.

4—It is also advantageous for the polymer which forms the DPs not toinduce an immune response (immunogenic).

5—The DPs are expected to allow the controlled and sustained release ofthe AP.

6—Lastly, it is also preferable for the DPs to be obtainable by aprocess which does not denature the AP. Thus, the use of organicsolvents and/or high temperatures or drastic pH changes should be ruledout.

Another essential aim of the invention is to provide lipid-based DPswhich are stable on storage, irrespective of the external medium, i.e.which retain their physical integrity equally well in a continuous phaseformed of water, formed of an aqueous solution or formed of an organicsolvent, such that they should not have a tendency to coalesce.

Another essential aim of the invention is to provide lipid-based DPswhich have a controllable and adjustable average particle size.

Another essential aim of the invention is to provide DPs which aresimple to prepare (non-corrosive pH), stable at any pH between 2 and 13,and non-immunogenic.

Another essential aim of the invention is to provide lipid-based DPswhich can be made industrially and in a cost-effective manner and whichcan be filled with either hydrophilic or lipophilic AP to high fillinglevels.

Another essential aim of the invention is to provide a process for thepreparation of lipid-based DPs which can be used as AP vectors, the saidprocess needing to be cost-effective, simple to carry out,non-denaturing for the APs and also needing to allow fine control of theaverage particle size of the particles obtained.

Another essential aim of the invention is the use of the said particlesfor the preparation of medicaments (e.g. vaccines) and/or nutrients, inparticular for oral, nasal, vaginal, ocular, subcutaneous, intravenous,intramuscular, intradermal, intraperitoneal, intracerebral or parenteraladministration of active principles such as proteins, glycoproteins,peptides, polysaccharides, lipopolysaccharides, oligonucleotides andpolynucleotides.

Another essential aim of the invention is to provide a medicament of thetype having a system with sustained release of AP, which isbiocompatible and which affords high availability of the AP.

The aims relating to the products, inter alia, are achieved by thepresent invention, which relates to particles, in particular for thedelivery of active principle(s), of the type based on microparticleswhich can be used as active principle (AP) vectors, characterized:

*α* in that they each have a cohesive structure made ofphysicochemically stable and integral composite gel,

*β* in that their cohesion is derived from the presence of the followingthree compounds:

(I) oil,

(II) an aqueous phase,

(III) and at least one linear, non-crosslinked, synthetic copolyaminoacid comprising at least two different types of amino acid comonomers:hydrophilic AA_(i) and hydrophobic AA₀,

*γ* and in that they have an average size which is controllable andadjustable over a range less than or equal to 500 μm, preferably to 200μm, and even more preferably between 0.05 and 100 μm.

It is to the Applicant's credit to have developed these partiallylipidic microparticles by, on the one hand, selecting specificconstituent products, namely oil, water and specific copolyamino acid,and, on the other hand, developing a procedure for obtaining the saidmicroparticles which leads, entirely surprisingly and unexpectedly, to acolloidal suspension of well-differentiated and extremely stablecohesive particles.

The stability is indeed one of the most surprising of the novelcharacteristics of the particles according to the invention, since theyretain their integrity and their differentiated nature both in a liquidmedium, irrespective of the pH of the medium, and in the form oflyophilysate. In other words, this means that they can be isolated byfiltration or decantation (e.g. by centrifugation), without coalescing.They thus form species of composite gel type, which have their ownidentity and existence, irrespective of the medium in which they arestored.

For the purposes of the present invention, the term “composite gel”denotes or refers to a notion of a physical gel based on water, oil andpolymer.

The DPs according to the invention are, moreover, biovectors that arecapable of including any active principle, irrespective of itshydrophilic or hydrophobic nature, and of delivering it inside the body.These DPs are biovectors which are all the more suitable:

since they are non-immunogenic, biodegradable and free of toxicstructuring and/or manufacturing products, even in trace amounts(solvents, glutaraldehyde),

and since their size (particle size) can be controlled over a widerange.

In conclusion, DPs are stable and cohesive microparticles. They do notcoalesce, in contrast with droplets of emulsion.

DETAILED DESCRIPTION OF THE INVENTION

The morphology of these cohesive DPs according to the invention is seenclearly in the scanning electron microscopy (SEM) photographs given inthe attached FIGS. 1 and 2. The description of these SEM photographs isrepeated below, in particular in the examples. However, it can alreadybe noted that these composite gel DPs are more or less spherical inshape and of smooth appearance. They are well differentiated from eachother.

As regards their structure, it is possible to say that it is neither aleaflet arrangement of the type found in liposomes, nor is it simpledroplets of heterogeneous phase of an O/W or W/O emulsion.

In the absence of tangible elements to characterize their structure, theApplicant has been able to define these composite gel DPs by means ofparticular functional features and, more specifically, behavioralfeatures of these DPs from discriminating technical evaluation tests.

Thus, the *α* characteristics of the DPs targeted above are reflected byat least one of the following functional properties:

α₁—absence of coalescence of the microparticles after a lyophilizationtreatment, this non-coalescence being reflected by a conservation of theparticle size distribution, in particular of the D[4.3] in a proportionof ±20%, after rehydration according to a test α₁;

α₂—absence of coalescence on centrifugation, reflected by a conservationof the particle size distribution, and in particular of the D[4.3] in aproportion of ±20%, according to a test α₂;

α₃—resistance to pH variations, reflected by a conservation of theparticle size distribution of the microparticles and, in particular, ofthe D[4.3] in a proportion of ±20%, after exposure to pHs of 3 and 13,according to a test α₃;

α₄—absence of coalescence in dispersion in a buffered aqueous solution,reflected by a conservation of the particle size distribution of themicroparticles, in particular of their D[4.3] in a proportion of ±20%,for storage times of greater than or equal to 3, 9 and 18 months attemperatures of 37° C., ambient and 4° C. respectively, according to atest α₄.

The tests α₁, α₂, α₃, and α₄ mentioned above are useful as definingstandards in the context of the present account. These tests are definedin detail below.

Test α₁ of DP stability to lyophilization/rehydration:

50 ml of a DP suspension at 0.2% by weight, calculated on the basis ofthe mass of polymer forming the DP, are prepared in a 500 mlround-bottomed flask. The buffered saline aqueous phase used consists of0.01 M PBS (phosphate buffer, pH 7.4 at 25° C.).

This solution is then frozen in liquid nitrogen before being lyophilizedusing a CHRIST-ALPHA1-4 brand lyophilization device, by subjecting thesample to a pressure of 20 Pa and a temperature of −52° C. for 48 h.

The lyophilizate thus obtained is then hydrated by placing it in thepresence of a volume of aqueous phase identical to that used to make thesuspension and representing 5 ml.

The particle size distribution of the DPs thus rehydrated is measured bylaser scattering using a COULTER LS 130 machine according to aFRAUNHOFER calculation model with “PIDS”. This allows the D[4.3] to beobtained.

The same particle size distribution measurement with determination ofthe D[4.3] is carried out on the DP suspension prepared above and theD[4.3] values obtained with and without lyophilization/rehydration arecompared.

Test α₂ of DP stability to centrifugation:

A DP suspension is prepared in the same way as in the text α₁ describedabove, except that the DP concentration in the suspension is 2% byweight instead of 0.2%.

A sample of 1.5 ml of the abovementioned suspension is centrifuged usinga SIGMA 3K30 brand centrifuge for 10 min at 60,000 g.

The sample is stirred manually in order to resuspend the DPs aftercentrifugation.

The particle size measurements (D[4.3]) are carried out as described intest α₁ above. The D[4.3] values obtained before and aftercentrifugation are compared.

Test α₃ of DP resistance to pH:

0.5 ml of DP suspension at 2% (concentration expressed in the same wayas that given in test α₁) is introduced into 5 ml of an HCl solutionwith a titer of 10⁻² N (pH 2).

This mixture is stored for one day at ambient temperature.

The suspension is neutralized by introduction of 50 μl of 1 N sodiumhydroxide. This mixture is made up to 10 ml with phosphate-bufferedsaline (PBS) solution, pH 7.4.

The D[4.3] particle size measurements are carried out on suspensionswhich have and have not had their pH lowered to a value of 2. The D[4.3]values obtained in the two cases are compared.

Exactly the same operation is carried out, but this time introducing 0.5ml of DP suspension at 2% (concentration expressed in the same way asthat given in test α₁) in 5 ml of 10⁻¹ N sodium hydroxide. The mixtureis neutralized by addition of 0.5 ml of 1 N HCl. It is made up to 10 mlwith isotonic phosphate-buffered saline (PBS) solution, pH 7.4.

The same measurements and the same particle size comparisons of D[4.3]are carried out.

Test α₄ of DP stability on storage:

A DP suspension is prepared which is identical in all respects to theone prepared in accordance with test α₂ described above.

Samples of this suspension (2 ml) are stored at temperatures of 2, 4 and37° C. and at ambient temperature.

The particle size distribution and, in particular, the D[4.3] (samemethod of measurement as for the tests α₁ to α₃ above) are monitored asa function of the storage time.

The D[4.3] values obtained after different storage times under thedifferent temperature conditions are compared with a D[4.3] referencemeasured on the suspension just after it has been prepared (t=0).

The storage time thresholds with conservation of the particle sizedistribution of the DPs are set arbitrarily as indicated below:

greater than or equal to three months at 37° C.;

greater than or equal to nine months at ambient temperature;

greater than or equal to eighteen months at 4° C.

Preferably, the composite gel DPs according to the invention have atleast two of the functional characteristics α₁, α₂, α₃ and α₄, and evenmore preferably α₁, α₂ and α₃.

These characteristics are illustrated below in the examples.

As indicated above, the selection of the copolyamino acid is one of thecrucial parameters of the invention. Thus, in accordance with apreferred characteristic, the copolyamino acid (III) has:

comonomers AA_(i) chosen from the following group of amino acids:glutamic acid, aspartic acid, ornithine, arginine, lysine, asparagine,histidine and mixtures thereof,

as well as comonomers AA_(o) chosen from the following group of aminoacids: leucine, tyrosine, phenylalanine, valine, cystine, isoleucine andmixtures thereof.,

Even more preferably, the copolyamino acid (III) comprises a comonomerAA_(i) formed of glutamic acid and/or aspartic acid, as well as amonomer AA_(o) formed of leucine and/or isoleucine and/or tyrosineand/or phenylalanine.

The linear copolyamino acids (III) according to the invention can be ofrandom structure or of diblock, triblock or multiblock structure, thediblock structure being preferred for reasons of non-immunogenicity.

The term random structure is understood to refer to a copolymer obtainedfrom comonomers having different reactivity ratios, which implies thatthe composition of these so-called random copolymers varies as afunction of the degree of conversion.

An example of a copolyamino acid (III) which is more specifically suitedto the invention is Leu/Glu, and in particular the one with a 50/50assay.

Limiting the number of comonomers to two amino acids, an AA₀ and anAA_(i), minimizes the immunogenicity of the DPs. This is a considerableadvantage of this preferred embodiment of the invention.

Besides their preferred linear structure containing α-peptide chains,the copolyamino acids (III) have, as another characteristic, a highmolar mass M_(W), i.e. greater than or equal to 5000 D, preferablybetween 8000 and 100,000 D. More preferably, the molar mass M_(W) of thecopolyamino acids (III) is selected as a function of their nature:multiblock or random. In the case of multiblock, in particular diblock,polyamino acids (III), the preferred ones are those with a mass M_(W) ofgreater than or equal to 5000 D, preferably between 3000 and 100,000 Dand even more preferably between 5000 and 20,000 D. As regards thecopolyamino acids (III) of random type, those with an M_(w) of greaterthan or equal to 50,000 D, preferably between 10,000 and 300,000 D andeven more preferably between 10,000 and 100,000 D, are more preferablyselected.

In fact, these copolyamino acids (III) are amphiphilic copolymers formedof a first type of monomers AA₀, which is a neutral hydrophobic aminoacid, and of at least a second type of comonomers AA_(i), which is anamino acid having a side chain of carboxyl functionality (Glu/Asp) whichcan be ionized at physiological pHs which do not denature proteins.These amphiphilic AAPs (III) can interact both with hydrophobicsubstances and with hydrophilic substances, which gives them noteworthyproperties as surfactants or dispersants. They thus participate in thesurprising effect by which the DPs acquire and conserve the quality ofdefinite stable bodies.

As regards the availability of the APs (III), it should be pointed outthat many techniques exist for the synthesis of block or random α-aminoacid polymers or of multiple-chain polymers or alternatively of polymerscontaining a determined amino acid sequence (cf. Encyclopedia ofPolymers Science and Engeneery [sic] (vol. 12, page 786, Ivan WILEY &Sons).

Many amino acid derivatives and peptides have been used as monomers forthe preparation of polyamino acids. However, the monomers mostfrequently used are N-carboxy-α-amino acid anhydrides, the preparationof which is given, for example, in Biopolymers, 15, 1869 (1976). Thetechniques for polymerizing these monomers are known to those skilled inthe art and are detailed in the book by H. R. KRICHELDORF“α-Aminoacid-N-Carboxy Anhydrides and Related Heterocycles” SpringerVerlag (1987)

The synthetic techniques generally involve protecting the reactivefunctions of the amino acids with ionizable side chains, in order forthem not to interfere during the polymerization step. Consequently, adeprotection step is necessary in order to reestablish the functionalityof the ionizable side chains of the polymer. Mention may be made, forexample, of processes of deprotection by saponification of methyl esters(STAHMAN et al.; J. Biol. Chem., 197, 771 (1952); KYOWA HAKKO, FR 2 152582) or debenzylation [BLOUT et al.; J. Amer. Chem. Soc., 80, 4631 (1858[sic])].

Now, as regards the oil (I), it may be indicated that, for the purposesof the invention, an oil denotes a substance which is liquid at ambienttemperature and which, in addition, is immiscible or only sparinglymiscible with water. Fatty substances which are liquid at ambienttemperature satisfy this definition. This is likewise the case forsilicone oils.

Thus, in accordance with a preferred characteristic of the invention,the oil (I) is formed of one or more fatty compounds selected from thefollowing group:

medium-chain fatty ester acid triglyceride(s) of animal, plant orsynthetic origin,

paraffin(s),

polysiloxane oil(s),

fatty acids of animal or plant origin,

fatty acids, esters thereof and/or salts thereof.

As examples of oils which are particularly suitable for the DPsaccording to the invention, mention may be made of

triglyceride oils such as coconut oil (such as the one sold under thebrand name “MYGLIOL®” by the company DYNAMIT NOBEL,

corn oil,

paraffin,

olive oil,

isopropyl palmitate,

butyl stearate,

ethyl oleate,

eicosapentanoic docosahexanoic acid esters,

alkyl benzoate,

eicosapentanoic docosahexanoic acids,

eicosapentanoic docosahexanoic acid triglycerides,

caprylic acid triglycerides,

silicone oil.

The oil (I) can consist of only one type of fatty substance which isliquid at ambient temperature or of a mixture of several of these.

According to a variant, the oil(s) (I) may or may not be fractionated.Fractionation makes it possible to remove certain fatty acid fractions,so as to modify the melting point and the viscosity.

As regards, more specifically, the melting point, it can be pointed out,in order to establish the ideas, that the melting point of the oil (I)is less than or equal to 50° C., preferably to 40° C. and, morepreferably, to 35° C.

The viscosity of the oil (I) at 25° C. is, in practice, between 10 mPa.sand 3000 mPa.s.

In quantitative terms, the oil (I) represents from 9 to 90% by weight,preferably from 20 to 80% by weight and even more preferably from 40 to60% by weight, of the DPs.

As regards the aqueous phase (II) of the lipid microparticles consideredhere, this preferably consists of a saline solution which willadvantageously be buffered, so as to have a pH of between 5 and 9,preferably between 6 and 8 and even more preferably of about 7.4.

The solutes in this solution are salts such as, for example, NaCl.

Advantageously, the molarity of the saline solution is between 10⁻⁴ Mand 1 M, preferably between 10⁻² M and 0.5 M approximately.

The buffers which may be used are, for example, the following:phosphate, phthalate, borate, etc.

The fact that the aqueous phase is preferably formed of a salinesolution is not exclusive of the variant in which water, advantageouslydeionized water, is involved.

The preferred lipid microparticles according to the invention are thosecharacterized by the following composition:

(I) medium-chain fatty ester acid triglyceride(s), coconut oil beingparticularly preferred,

(II) deionized water or buffered saline solution, the pH of this aqueousphase being between 6 and 8,

(III) copolyamino acid of Leu/Glu type, preferably 50/50.

Beyond the structural and functional characteristics of the DPs, such asthose which have been mentioned above, the subject of the presentinvention is also a process for the preparation of microparticles, inparticular such as those defined above, characterized in that itcomprises, essentially, the following successive or non-successivesteps:

-a- preparation of a gel from the aqueous phase (II) and of at least onecopolyamino acid (III), (II) and (III) being as defined in thedescription,

-b- placing the gel obtained from step -a- in contact with oil (I), asdefined in the description,

-c- stirring the gel+(I) mixture leading to a dispersion ofmicroparticles in an oily or aqueous continuous phase (I),

-d- optional separation of the microparticles and of the oily or aqueouscontinuous phase, preferably by centrifugation,

-e- optional redispersion of the microparticles collected after step -e-in a storage liquid,

-f- optional lyophilization treatment of the microparticles from step-d-, which are or are not redispersed.

One of the foundations of this process and of the microparticles arisingtherefrom concerns step -a- for the preparation of a gel derived fromthe combination of the copolyamino acid (III) and of the aqueous phase(II). This more specifically involves mixing (II) with (III) inproportions such that (III) represents from 2 to 50%, preferably from 5to 30%, by weight of the gel (III)+(II). This mixing is carried out withstirring using any suitable stirring means known per se. This means canbe, for example, a vortex type device or alternatively a rotor-stator,magnetic bar, ultrasound or high-pressure homogenizer type device.

Advantageously, this step -a- is combined with a treatment for removalof the foam formed during the stirring. Such a removal can be carriedout, for example, by centrifugation.

The gel obtained in step -a- is then placed in contact with all or someof the oil (I) (step -b-), the mixture thus formed then being subjectedto stirring (step -c-) with stirring means of the same type as thosementioned for step -a-. This step -c- corresponds to the step for theformation of the lipid microparticles.

The introduction of the oil (I) into the mixture from step -b- can becarried out one or more times during step -c-.

It is clear that the stirring conditions are determining factors for theformation of the DPs in step -c-, in particular with regard to thenature of the homogeneous phase containing the DPs: water or oil.

Thus, according to a preferred embodiment of the invention, leading tocomposite gel DPs suspended in an aqueous phase, this stirring -c- iscarried out using a rotor/stator device and working at a stirring speedof between 1000 and 40,000 rpm, preferably 5000 and 25,000 rpm.

According to a variant of this preferred embodiment, which is aimed atobtaining DPs suspended in an oily phase, the suspension is enrichedwith oil.

At the end of step -c-, it is possible to obtain an interestingintermediate product, formed of a concentrated dispersion of DPs inwater or a suspension of DPs in oil. In the latter case, the excess oilcan be removed and a concentrated DP sedimentate is thus obtained.

The subject of the present invention is thus also this concentrateddispersion and this concentrated sedimentate of DPs in water, which canbe considered as intermediate products.

These intermediate products are characterized by:

an average concentration of copolyamino acids (III) ranging from 1% to50% by weight, preferably from 2% to 40% by weight, this concentrationbeing established, even more preferably, from 3% to 30% by weight;

a concentration of oil (I) representing from 9 to 90% by weight,preferably from 20 to 80% by weight and even more preferably from 40 to60% by weight;

a concentration of aqueous phase of from 5 to 90% by weight, preferablyfrom 10 to 70% by weight, even more preferably from 30 to 50% by weight;

the above concentrations being expressed relative to the total mass ofthe concentrated DP dispersion or sedimentate.

The relative amounts of oil (I) and of aqueous phase (II) will overrideeach other depending on the hydrophilic or hydrophobic nature intendedfor the DPs. In the biovector application, this nature will depend onthe active principle to be delivered.

To summarize, it can thus be indicated that the microparticles accordingto the invention can be defined by the following quantitativecharacteristics, according to which their constituents (I), (II) and(III) are present in the proportions below, expressed as % by weightrelative to (I)+(II)+(III):

(I) 9 to 90, preferably 20 to 80 and even more preferably 40 to 60,

(II) 5 to 90, preferably 10 to 70 and even more preferably 30 to 50,

(III) 1 to 50, preferably 2 to 20 and even more preferably 3 to 10.

The microparticles can be recovered (step -d-) by any suitable knownmeans, centrifugation or decantation being examples of such means.

The medium for dispersing the DPs recovered in step -d- can be filtereddeionized water or a buffered solid solution, to which at least onebacteriostatic agent may be added.

Advantageously, the temperature at which steps -a- to -c-, or even -d-to -f- of the process according to the invention are carried out isbetween 4° C. and 60° C. Ambient temperature is particularlyappropriate, this preferably being between 10 and 35° C.

As emerges from the text hereinabove, the process according to theinvention allows the spontaneous generation of lipid microparticles bymeans of a noteworthy, simple, cost-effective and thus industriallyfeasible procedure. It will also be noted that the process according tothe invention is unquestionably and invariably safe, since it does notenvisage using toxic solvents or reagents.

The important parameters of the process according to the invention are,in particular, the nature of the oil (I), the composition of thecopolyamino acid (III) and its concentration, the concentration of thesaline solution (II), the stirring conditions and the pH of the reactionmedium.

A person skilled in the art is capable of relatively easily controllingall of these conditions for the preparation of the DPs according to theinvention. In particular, it is entirely within his or her ability tocontrol and adjust the particle size of the lipid microparticles byvarying the stirring conditions and the ratio of the viscosities of thephases present, inter alia.

Since one of the most noteworthy applications of the particles accordingto the invention is the protected delivery of active principles (APs)into the human or animal body, it is worthwhile, at this stage in theaccount, providing details on the technique(s) for including the AP intothe DPs.

One of the preferred inclusion techniques in accordance with theinvention consists in dissolving or suspending the said AP:

in the oil (I),

and/or in the aqueous phase (II),

and/or in the polyamino acid (III),

and/or in the gel from step -a-.

The AP to be included in the DPs can be in solid form or in the form ofa solution or a dispersion.

The active principle which can be included or incorporated into the DPsaccording to the invention can be medicinal and/or nutritional. When itis medicinal, the AP is preferably chosen from:

proteins and/or peptides, among which those most preferably selectedare: hemoglobins, cytochromes, albumins, interferons, antigens,antibodies, calatonin, erythropoietin, insulin, growth hormones, factorIX, interleukin or mixtures thereof,

polysaccharides, heparin being more particularly selected,

nucleic acids and, preferably, RNA and/or DNA oligonucleotides,

and mixtures thereof.

The APs, which can be classified in the category of medicaments andwhich can be delivered by the particles according to the invention, arevaccines.

As examples of nutritional APs, mention may be made of vitamins, aminoacids and trace elements.

INDUSTRIAL APPLICATION

According to another of its aspects, the invention is also directedtowards use of DPs filled with AP, for the manufacture of medicaments,in particular of the type including a system with controlled release ofAP.

Lastly, the present invention relates to the medicaments and thepharmaceutical and nutritional specialties comprising the microparticlesfilled with AP, as described above.

The pharmaceutical specialties concerned are, in particular, thosepreferably for oral, nasal, vaginal, ocular, subcutaneous, intravenous,intramuscular, intradermal, intraperitoneal, intracerebral or parenteraladministration.

The applications of the invention are not limited to vectorization, tothe delivery of an AP of medicinal or nutritional nature, since it isentirely conceivable that the AP, which can be included or incorporatedinto the DPs, might be a cosmetic or plant-protection product.

The cosmetic applications which can be envisaged are, for example,compositions which can be applied transdermally.

The plant-protection products concerned can be, for example, herbicidesand/or fungicides and/or bactericides and/or virucides and/orinsecticides, inter alia.

The subject of the present invention is also the plant-protection andcosmetic compositions comprising DPs filled with AP of the type referredto above.

The examples which follow will give a clearer understanding of theinvention in its various product/process/application aspects. Theseexamples illustrate the preparation of DP microparticles, which are orare not filled with active principles and based on oil (I), water (II)and AAP (III). These examples also show the structure characteristics,as well as the properties of the said microparticles.

The illustration of the examples is given by FIGS. 1 to 9 describedbelow.

DESCRIPTION OF THE FIGURES

FIG. 1 is a scanning electron microscopy (SEM) photograph of thecomposite gel DPs prepared in Example 4, magnification: 1500×.

FIG. 2 is a scanning electron microscopy (SEM) photograph of thecomposite gel DPs prepared in Example 5, magnification: 3200×.

FIG. 3 is a histogram of voluminal distribution of composite gel DPs:differential volume (V) in % as a function of the diameter (D) in μm(Example 4).

FIG. 4 is a histogram of voluminal distribution of lyophilized andrehydrated composite gel DPs: differential volume (V) in % as a functionof the diameter (D) in μm (Example 4).

FIG. 5 is a histogram of voluminal distribution of composite gel DPs:differential volume (V) in % as a function of the diameter (D) in μm(Example 5).

FIG. 6 is a histogram of voluminal distribution of composite gel DPs:differential volume (V) in % as a function of the diameter (D) in μm(Example 6).

FIG. 7 is a histogram of voluminal distribution of composite gel DPs(differential volume (V) in % as a function of the diameter (D) in μm):

curve 1: before lyophilization/rehydration,

curve 2: after lyophilization/rehydration (Example 7).

FIG. 8 represents a histogram giving the average diameters D[4.3] byvolume and the standard deviations (SD) measured for the DP particlesize distributions according to Tests 1 to 14 of Example 9.

FIG. 9 is a graph giving the percentage R of release of theAP=cytochrome as a function of the residence time T in hours (Example11).

EXAMPLES Example 1 50/50 Poly(LEUCINE-SODIUM GLUTAMATE) Random Synthesis

STEP 1): COPOLYMERIZATION OF NCA-LEU AND NCA-GLU(OMe): 50/50POLY(LEU-CO-GLU(OMe)):

15.0 g of methyl glutamate N-carboxyanhydride (NCA-Glu(OMe): 0.08 mol)and 12.5 g of leucine N-carboxyanhydride (NCA-Leu: 0.08 mol) areintroduced, under a stream of nitrogen, into a 1 l reactor fitted with aglass stirrer, a nitrogen inlet and an outlet connected to a bubbler.381 ml of dioxane are added and the reaction medium is brought to 40° C.

After dissolving the NCA, 24 ml of water are added, followed by 0.22 mlof triethylamine (i.e. 1 mol % relative to the NCA). The polymerizationis monitored by IR by observing the disappearance of the carbonyl bandsat 1860 and 1790 cm⁻¹. The polymerization time ranges between 1.5 h and3 h depending on the composition of the monomers. After the bands havetotally disappeared, the reaction medium is diluted with 380 ml ofdioxane and then homogenized for 3 h at ambient temperature. Thecopolymer is recovered by precipitation from 5 l of water with vigorousstirring. The product is filtered off and dried at 50° C. under vacuumfor 12 h.

The mass of copolymer obtained is 18.4 g, i.e. a weight yield of 90%.

¹H NMR (d-trifluoroacetic acid): 0.85 ppm (CH₃-Leu, 6H*0.5); 1.58 (CH₂and CHMe₂ Leu, 3H*0.5); 2.10 and 2.22 (CH₂-Glu, 2H*0.5); 2.58 (CH₂-Glu;2H*0.5); 3.75 (CH₃-Glu, 3H*0.5); 4.62 (NCHCO-Leu, 1H*0.5); 4.70(NCHCO-Glu, 1H*0.5). Reduced viscosity (0.5 g/dl in trifluoroaceticacid) at 25° C.=2.2 dl/g.

STEP 2): HYDROLYSIS OF THE METHYL ESTER OF 50/50 POLY(LEU-CO-GLU(OMe)):

The copolymer obtained above (17.7 g) is placed in a reactor into whichare added 354 ml of trifluoroacetic acid. The reaction medium is broughtto 40° C. with stirring. When the copolymer has totally dissolved, 354ml of water are added portionwise. The reaction medium is kept stirringfor 48 h. The polymer is recovered by precipitation from 5 l of water.After filtration, it is again suspended and stirred in water for 0.5 h,and then filtered off and drained. The purification is carried out bydialysis in water. 15.9 g (95%) yield. ¹H NMR (d-trifluoroacetic acid):identical to the starting polymers except that the signal at 3.75(CH₃-Glu) is greatly reduced or absent. In the present case, the levelof residual esters is less than 1% relative to the glutamate monomers.Reduced viscosity (0.5 g/dl in trifluoroacetic acid) at 25° C.=0.95dl/g.

Example 2 Synthesis of the 50/50 Poly(LEU-B-GLU(ONa)) Diblock

15.0 g of NCA-Glu(OMe) (0.08 mol) and 180 ml of dioxane are introducedinto a 1 1 reactor with stirring. After dissolution, 180 ml of tolueneare added and the medium is brought to 60° C. The IR spectrum of thesolution is acquired, after which 0.156 g of benzylamine (1.58 mol%/NCA) is added. The reaction medium rapidly becomes cloudy and, after40 minutes, the characteristic bands at 1860 and 1790 cm⁻¹ havedisappeared.

After one hour, a solution of 12.5 g of NCA-Leu (0.08 mol) in adioxane/toluene mixture (15 ml of each) is introduced. Stirring iscontinued for 18 h (this duration was not optimized). The carbonyl bandshave by then disappeared.

100 ml of dioxane are added and the reaction medium is homogenized for 1h. The copolymer is precipitated from 3 l of absolute ethanol withvigorous stirring. It is washed with 1 l of ethanol, filtered off,drained and finally dried at 50° C. after vacuum overnight.

The mass of product recovered is 19.5 g (yield=95%).

¹H NMR (d-trifluoroacetic acid): 0.85 ppm (CH₃-Leu, 6H*0.5); 1.58 (CH₂and CHMe₂ Leu, 3H*0.5); 2.10 and 2.22 (CH₂-Glu, 2H*0.5); 2.58 (CH₂-Glu;2H*0.5); 3.75 (CH₃-Glu, 3H*0.5); 4.62 (NCHCO-Leu, 1H*0.5); 4.70(NCHCO-Glu, 1H*0.5). Reduced viscosity (0.5 g/dl in trifluoroaceticacid) at 25° C.=0.62 dl/g.

The second step for hydrolysis of the methyl esters is identical to thatdescribed in Example 1, step 2. 95% yield. ¹H NMR (d-trifluoroaceticacid): identical to the starting polymers except that the signal at 3.75(CH₃-Glu) is greatly reduced or absent,. In the present case, the levelof residual esters is less than 1% relative to the glutamate monomers.Reduced viscosity (0.5 g/dl in trifluoroacetic acid) at 25° C.=0.55dl/g.

Example 3 Synthesis of the 25/50/12 Poly(GLU(ONa)-LEU-GLU(ONa)) Triblock

7.5 g of NCA-Glu(OMe) (0.04 mol) and 180 ml of dioxane are introducedinto a 1 l reactor with stirring. After dissolution, 180 ml of tolueneare added and the medium is brought to 60° C. The IR spectrum of thesolution is acquired, after which 0.156 g of benzylamine is added.

After the monomer has totally disappeared, a solution of 12.5 g ofNCA-Leu (0.08 mol) in a dioxane/toluene mixture (15 ml of each) isintroduced. Stirring is continued for 18 h. Next, a further 7.5 g ofNCA-Glu(OMe) (0.04 mol) are added and are allowed to react for 12 hours.100 ml of dioxane are added and the reaction medium is homogenized for 1h.

The copolymer is precipitated from 3 l of absolute ethanol with vigorousstirring. It is washed with 1 l of ethanol, filtered off, drained andfinally dried at 50° C. under vacuum overnight.

The mass of product recovered is 19.4 g (yield=95%).

¹H NMR (d-trifluoroacetic acid): 0.85 ppm (CH₃-Leu, 6H*0.5); 1.58 (CH₂and CHMe₂ Leu, 3H*0.5); 2.10 and 2.22 (CH₂-Glu, 2H*0.37); 2.58 (CH₂-Glu;2H*0.37); 3.75 (CH₃-Glu, 3H*0.37); 4.62 (NCHCO-Leu, 1H*0.5); 4.70(NCHCO-Glu, 1H*0.37). Reduced viscosity (0.5 g/dl in trifluoroaceticacid) at 25° C.=0.58 dl/g.

The second step for hydrolysis of the methyl esters is identical to thatdescribed in Example 1, step 2. ¹H NMR (d-trifluoroacetic acid):identical to the starting polymer except that the signal at 3.75(CH₃-Glu) is greatly reduced or absent. In the present case, the levelof residual esters is less than 1% relative to the glutamate monomers.Reduced viscosity (0.5 g/dl in trifluoroacetic acid) at 25° C.=0.38dl/g.

Example 4 Neutral Microparticles Prepared from Miglyol®, Water andPoly(LEUCINE-CO-SODIUM GLUATMATE) of 50/50 Composition and of RandomStructure

4.1—METHODOLOGY:

4.1.1—STEP a):

PREPARATION OF THE GEL:

50 mg of poly(leucine-CO-sodium glutamate) (abbreviated as Leu/Glu)lyophilizate, of

50/50 composition, of random structure, synthesized according to Example1 and with an M_(W) of 110,000, are introduced into a hemolysis tube.

The aqueous phase is added, typically 500 mg, which can be composed ofdeionized water or a buffered saline solution, for example 0.01 M PBS(phosphate buffer, pH 7.4 at 25° C.).

The polyamino acid lyophilizate is left to become hydrated for 2 hoursat ambient temperature.

The gel obtained is in the form of a viscous colloidal solution whichscatters in the blue region.

4.1.2—STEPS b) to e):

PREPARATION OF THE MICROPARTICLES:

(i) Process to obtain DPs in an oily continuous phase:

2 ml of the lipid phase composed of fractionated coconut oil, knownunder the trade name Miglyol® (DYNAMIT NOBEL), are introduced into thehemolysis tube containing the polyamino acid gel.

The mixture is stirred using a rotor/stator type homogenizer(ULTRA-TURRAX T8, TKA LABORTECHNIK). A milky-looking dispersion ofmicroparticles in a lipidic continuous phase is obtained.

The hemolysis tube containing the microparticle dispersion iscentrifuged. The supernatant phase, composed of excess Miglyol®, isseparated from the sedimentate by simply pouring it off. 1340 mg of oilare thus collected.

The sedimentate is redispersed in deionized water containing abacteriostat: Thimerosal® (50 μg/ml), or in a buffered saline solution,for example 0.01 M PBS (phosphate buffer, pH 7.4 at 25° C.) containingThimerosal® (50 μg/ml). The total volume of the dispersion obtained is5000 μl.

(II) Process to obtain DPs in an aqueous continuous phase:

400±2 μl of the lipid phase, composed of fractionated coconut oil knownunder the trade name Miglyol® (DYNAMIT NOBEL), are introduced into thehemolysis tube containing the polyamino acid gel.

The mixture is stirred with a rotor/stator type homogenizer(ULTRA-TURRAX T8, IKA LABORTECHNIK). A very dense milky dispersion ofmicroparticles in a continuous water phase is obtained.

The dispersion is then diluted in deionized water containing Thimerosal®(50 μg/ml), or in a buffered saline solution, for example 0.01 M PBS(phosphate buffer, pH 7.4 at 25° C.) containing Thimerosal® (50 μg/ml).The total volume of the dispersion obtained is 5000 μl.

4.2—CHARACTERIZATION OF THE MICROPARTICLES:

The overall composition of the microparticles before redispersion is:

4% polyamino acid,

53% Miglyol®,

43% water or saline solution.

The particle size distribution of the microparticles is measured bylaser scattering. The machine used is a COULTER LS 130 machine. Thecalculation model chosen is the FRAUNHOFER model with “PIDS”. Thevoluminal distribution histogram is given in FIG. 3. The profile ismonomodal, the average reference diameter D[4.3] is 2.8 μm with astandard deviation (SD) of 1.1 μm. Expressed differently, the particlesize distribution is as follows:

% of DP: 10.00 25.00 50.00 75.00 90.00 Size in μm less 5.165 3.876 2.7301.901 1.394 than:

The microparticles were observed by scanning electron microscopy (SEM)on a cold stage. The photographs are given [lacuna] FIG. 1.

The microparticles conserve their identity and their integrity in thesolvents, such as acetone, dimethyl sulfoxide and ethanol, and inaqueous media over the pH range between pH=2 and pH=13.

Test α₁: Lyophilization/rehydration:

The suspension, diluted in 50 ml, is lyophilized for 48 h. 529 mg ofmicroparticle lyophilizate are recovered. Rehydration of all thelyophilizate in 5 ml of PBS leads spontaneously to a suspension ofmicroparticles having a monomodal particle size profile similar to thatof the microparticles before lyophilization (cf.

FIG. 4). The average voluminal diameter, D[4.3], is 2.9 μm and thestandard deviation is 2 μm, i.e. a Δ of 3.5% relative to the referenceD[4.3].

Test α₁: Results:

% of DP: 10 25 50 75 90 Size in μm less 0.907 1.385 2.322 3.873 5.816than:

Test α₂:

A D[4.3] of 3 μm is found with an estimation of the standard deviationof 2 μm, i.e. a Δ of 7.1% relative to the reference D[4.3].

Test α₃:

at pH 2, a D[4.3] of 3.9 μm with an SD of 2.9 μm is measured, i.e. a Δof 3.5% relative to the reference D[4.3],

at pH 13, a D[4.3] of 3.0 μm with an SD of 2.2 μm is measured, i.e. a Δof 7.1% relative to the reference D[4.3].

Test α₄:

The shape stability of the microparticles stored in aqueous or salinedispersion, at 4° C., 37° C. and at ambient temperature, is analyzed bymonitoring the particle size of the microparticles. The microparticlesare considered as being physically stable when the recorded change inthe average diameter D[4.3] does not exceed more than 20% of the initialD[4.3].

Test α₄: results:

Storage Ambient temperature 4° C. temperature 37° C. Stability in >1year aqueous dispersion Stability in >6 months >3 months salinedispersion

Example 5 Neutral Nanoparticles Prepared from Miglyol®, Water andPoly(LEUCINE-CO-SODIUM GLUTAMATE) of 50/50 Composition and of RandomStructure

5.1—METHODOLOGY:

5.1.1—STEP a):

PREPARATION OF THE GEL:

The process is performed according to Example 4.1.1, using a mass of 150mg of the polyamino acid mentioned above. The aqueous phase is thustypically 1350 mg.

5.1.2—STEP b) to e):

PREPARATION OF NANO-PARTICLES

1200 μl of Miglyol® are introduced into the test tube containing thepolyamino acid gel.

The mixture is stirred with a rotor/stator type homogenizer(ULTRA-TURRAX T8, IKA LABORTECHNIK). A concentrated, viscous aqueoussuspension of micro-capsules is thus obtained.

This suspension is diluted in a total volume of 15 ml of deionized wateror of phosphate-buffered isotonic solution at pH 7.4.

The dilute suspension of microparticles is placed in the reservoir of ahigh-pressure homogenizer such as the MICROFLUIDICS brand MicrofluidizerM-110S.

The dilute suspension of microparticles is homogenized for 10 min,applying an inlet pressure of about 5 bar to the pump.

A suspension of nanoparticles is thus collected on draining thereservoir.

5.2—CHARACTERIZATION OF THE NANOPARTICLES:

The overall composition of the nanoparticles before dilution is: 6%polyamino acid; 43% Miglyol®; 51% water or saline solution.

The particle size distribution of the microparticles is measured bylaser scattering. The machine used is a COULTER LS130 machine; thecalculation model chosen is the Psl O.M.D. model with “PIDS”. Thevoluminal distribution histogram is given in FIG. 5. The averagereference diameter D[4.3] is 0.420 μm with a standard deviation of 0.37μm.

% of DP: 10 25 50 75 90 Size in μm less 0.191 0.242 0.321 0.434 0.592than:

Test α₂:

A D[4.3] of 0.45 μm and an SD of 0.41 μm are obtained, i.e. a Δ of 7.1%relative to the reference D[4.3].

Example 6 Neutral Microparticles Prepared from Miglyol®, Water andPoly(LEUCINE-CO-SODIUM GLUTAMATE) of 50/50 Composition and of DiblockStructure

6.1—METHODOLOGY:

6.1.1—STEP a):

PREPARATION OF THE GEL:

146 mg of poly(leucine-co-sodium glutamate) (abbreviated as LEU/GLU)lyophilizate synthesized according to Example 2 are introduced into ahemolysis tube.

The aqueous phase is added, typically 702±0.2 mg, which can be composedof deionized water filtered to 0.2 μm, or a buffered saline solution,for example 0.01 M PBS (phosphate-buffered isotonic solution at pH 7.4at 25° C.).

The polyamino acid lyophilizate is left to become hydrated.

The gel obtained is in the form of a viscous colloidal solution whichscatters strongly in the white region.

6.1.2—STEPS b) to e):

PREPARATION OF THE MICROCAPSULES:

The process is then performed as in Example 4.1.2(i). Aftercentrifugation, 1518 mg of oil are collected.

6.2—CHARACTERIZATION OF THE MICROCAPSULES:

The overall composition of the microparticles before redispersion is:12% polyamino acid; 31% Miglyol®; 57% water or saline solution.

The particle size distribution of the microparticles is measured bylaser scattering. The machine used is a COULTER LS130 machine; thecalculation model chosen is the FRAUNHOFER model with “PIDS”. Thevoluminal distribution histogram is given in FIG. 6. The profile ismonomodal, the average reference diameter D[4.3] is 1.9 μm with astandard deviation of 1.2 μm.

% of DP: 10 25 50 75 90 Size in μm less 0.801 1.096 1.605 2.433 3.678than:

The microparticles were observed by SEM on a cold stage. The photographsare given in FIG. 2.

Test α₄:

A Δ for the D[4.3] of less than or equal to 10%, relative to thereference D[4.3], is obtained.

Test α₄—Results:

Storage Ambient temperature 4° C. temperature 37° C. Stability in ≧18months ≧12 months ≧3 months saline dispersion

Example 7 Neutral Microparticles Prepared from Miglyol®, Water andPoly(LEUCINE-CO-SODIUM GLUTAMATE) of 30/70 Composition and of RandomStructure

7.1—METHODOLOGY:

7.1.1—STEP a):

PREPARATION OF THE GEL:

100 mg of poly(leucine-co-sodium glutamate) (abbreviated as LEU/GLU)lyophilizate of 30/70 composition, synthesized according to theprocedure given in Example 1, changing the monomer ratio, are introducedinto a hemolysis tube.

The aqueous phase is added, typically 400 mg, of 0.01 M PBS(phosphate-buffered isotonic solution at pH 7.4 at 25° C.).

The polyamino acid lyophilizate is left to become hydrated.

The gel obtained is in the form of a viscous colloidal solution whichscatters in the blue region.

7.1.2—STEP b) to e):

PREPARATION OF THE MICROCAPSULES:

The process is performed as described in Example 4.1.2(i). Aftercentrifugation, 1620 mg of oil are collected.

7.2—CHARACTERIZATION OF THE MICROCAPSULES:

The overall composition of the microparticles before redispersion is:12% polyamino acid; 38% Miglyol®; 50% saline solution.

The particle size distribution of the microparticles, measured by laserscattering under the standard conditions stipulated in Example 4, givesan average reference diameter D[4.3] of 2.9 μm with a standard deviationof 1.5 μm (cf. FIG. 8, curve 1).

LYOPHILIZATION/REHYDRATION:

Test α₁:

The dispersion of microparticles, thus obtained and characterized, islyophilized for 48 hours. 212 mg of microparticle lyophilizate arerecovered. Rehydration of all of this lyophilizate in 5 ml of PBS leadsspontaneously to a microparticle suspension having a particle sizeprofile which is virtually superimposable on the one before thelyophilization step. The D[4.3] is 3.0 μm with a standard deviation of1.8 μm (cf. FIG. 7, curve 2), i.e. a Δ D[4.3], relative to the referenceD[4.3], of 3.4%.

Example 8 Neutral Microparticles Prepared from Silicone Oil, Water andPoly(LEUCINE-CO-SODIUM GLUTAMATE) of 30/70 Composition and of RandomStructure

8.1—METHODOLOGY:

8.1.1—STEP a):

PREPARATION OF THE GEL:

50 mg of poly(leucine-co-sodium glutamate) (abbreviated as LEU/GLU)lyophilizate of 30/70 composition, synthesized according to theprocedure given in Example 1, changing the monomer ratio, are introducedinto a hemolysis tube.

The aqueous phase is added, typically 450 mg, of 0.01 M PBS(phosphate-buffered isotonic solution at pH 7.4 at 25° C.).

The polyamino acid lyophilizate is left to become hydrated. v

The gel obtained is in the form of a viscous colloidal solution whichscatters in the blue region.

8.1.2—STEP b) to e):

PREPARATION OF THE MICROCAPSULES:

2000 μl of silicone oil of reference code Rhodorsil® 47V20 areintroduced into the hemolysis tube containing the polyamino acid gel.

The mixture is stirred with a rotor/ stator type homogenizer(ULTRA-TURRAX T8, IKA LABORTECHNIK). A milky-looking dispersion ofmicroparticles in an oily continuous phase is obtained.

The hemolysis tube containing the microparticle dispersion iscentrifuged (20 min at 3500 rpm). The supernatant phase, composed ofexcess silicone oil, is separated from the sedimentate by simply pouringit off; 921 mg of oil are thus collected.

The sedimentate is redispersed in deionized water containing Thimerosal®(50 μg/ml) or in a buffered saline solution, for example 0.01 M PBS(phosphate buffer, pH 7.4 at 25° C.) containing Thimerosal® (50 μg/ml).The total volume of the dispersion obtained is 5000 μl.

8.2—CHARACTERIZATION OF THE MICROCAPSULES:

The overall composition of the micro-particles before redispersion is:3.3% polyamino acid; 66.6% Rhodorsil®; 30.1% saline solution.

The particle size distribution of the microparticles, measured by laserscattering under the standard conditions stipulated in Example 4, givesan average diameter D[4.3] of 4.7 μm with a standard deviation of 1.7μm.

Example 9 Neutral Microparticles Prepared from Oils of Different Nature,Water and Poly(LEUCINE-CO-SODIUM GLUTAMATE) of 50/50 Composition and ofRandom Structure

9.1—METHODOLOGY:

The process is performed as indicated in Example 8 and the followingoils are used:

TEST COMMON NAME TRADE NAME SUPPLIER 1 oleyl alcohol NOVOL CRODA 2eicosapentanoic INCROMEGA TG CRODA docosahexanoic acid triglyceride 3caprylic capric acid MIGLYOL HULS triglyceride 4 caprylic acidtriglyceride TRICAPRYLIN C8 SIGMA 5 triglyceride of caprylic CRODAMOLGTCC CRODA acid triglycerides [sic] 6 alkyl benzoate CRODAMOL AB CRODA 7isopropyl palmitate CRODAMOL IPP CRODA 8 butyl stearate CRODAMOL BSCRODA 9 eicosapentanoic INCROMEGA E CRODA docosahexanoic acid esters 10ethyl oleate ETHYL OLEATE CRODA 11 liquid paraffin C16/C21 MOREL/fraction SALABE 12 silicone oil RHODORSIL RHONE 47V100 POULENC 13 oliveoil REFINED OLIVE CRODA OIL 14 eicosapentanoic INCROMEGA F CRODAdocosahexanoic acids

9.2—CHARACTERIZATION OF THE MICROCAPSULES:

The overall composition of the micro-particles, before redispersion,varies depending on the lipid phases used, within the following ranges:3 to 4% polyamino acid; 64 to 69% oil; 28 to 32% water or salinesolution. Only the microparticles based on INCROMEGA E show a differentcomposition before redispersion: 5% polyamino acid; 51% oil; 44% wateror saline solution.

The particle size distributions of the microparticles are measured bylaser scattering. The machine used is a COULTER LS130 machine; thecalculation model chosen is the FRAUNHOFER model with “PIDS”. Theaverage voluminal diameters, D[4.3], and the standard deviationsobtained are given on the graph in FIG. 8.

Example 10 Neutral Microparticles Prepared from Miglyol®, Water andVarious Copoly(α-AMINO ACIDS) of Random Structure

10.1—METHODOLOGY:

The process is performed as mentioned in Example 4.1.1 and 4.1.2(i). Thecopoly(α-amino acids) used are those given in the table below. Thehydration time of the lyophilizate of polymer in the isotonic solution(0.01 M PBS buffered to pH 7.4) is 24 hours. The gels obtained havedifferent viscosities.

No. COPOLY(α-AMINO ACIDS) SUPPLIER 1 80/20 Poly(Glu; Phe) SIGMA 2 80/20Poly(Glu/Leu) SIGMA 3 50/50 Poly(Glu/Tyr) SIGMA 4 25/75 Poly(Glu/Leu)FLAMEL TECHNOLOGIES 5 50/50 Poly(Orn/Leu) SIGMA

10.2—CHARACTERIZATION OF THE MICROPARTICLES:

The overall composition of the micro-particles, before redispersion,varies depending on the polymers used, within the following ranges:

No. % WATER % MYGLIOL % POLYMER 1 20 77 3 2 33 62 5 3 51 43 6 4 39 56 55 27 68 5

The particle size distributions of the microparticles are measured bylaser scattering. The machine used is a COULTER LS130 machine; thecalculation model chosen is the FRAUNHOFER model with “PIDS”. Theaverage voluminal diameters, D[4.3], and the standard deviations (SD)obtained are given in the table below:

No. D[4.3] μm SD μm DISTRIBUTION PROFILE 1 5.5 3.6 monomodal 2 8.8 8.6bimodal 3 155.5 114 monomodal 4 5.9 3.9 monomodal 5 9.7 11.0 bimodal

The microparticles of batches 1 to 5 satisfy test α₂:

Example 11 Microparticles Containing Cytochrome, Prepared from Miglyol®,Water and Poly(LEUCINE-CO-SODIUM GLUTAMATE) of 50/50 Composition and ofRandom Structure—DIRECT Encapsulation

11.1—METHODOLOGY:

Three batches of microcapsules were manufactured according to theidentical operating conditions described below.

11.1.1—STEP a):

PREPARATION OF THE GEL:

1.5 ml of a stock solution of horse heart cytochrome C (ref. C2506SIGMA) at 60 mg/ml in 0.01 M isotonic phosphate buffer (PBS), pH 7.4,are prepared.

50±0.2 mg of poly(leucine-co-sodium glutamate) (abbreviated as LEU/GLU)lyophilizate of 50/50 composition, of random structure and with an M_(W)of 110,000, are introduced into a hemolysis tube.

450 mg of the cytochrome stock solution are added.

The polyamino acid lyophilizate is left to become hydrated.

The gel obtained is in the form of a viscous orange/red colloidalsolution.

11.1.2—STEPS b) to e):

PREPARATION OF THE MICROCAPSULES ACCORDING TO THE PROCEDURE OF EXAMPLE4.1.2(i):

2000 μl of Miglyol® (DYNAMIT NOBEL) are introduced into the hemolysistube containing the polyamino acid gel and cytochrome.

The mixture is stirred with a rotor/stator type homogenizer(ULTRA-TURRAX T8, IKA LABORTECHNIK). A milky-looking dispersion ofmicroparticles in a lipidic continuous phase is obtained.

The hemolysis tube containing the microparticle dispersion iscentrifuged. The supernatant phase, composed of excess Miglyol®, isseparated from the sedimentate by simply pouring it off. 1577 mg of oilare thus collected.

The sedimentate is redispersed in PBS filtered to 0.22 μm containingThimerosal® (50 μg/ml). The total volume of the dispersion obtained is20 ml.

Storage of the microparticle dispersion in a ventilated chamberconditioned to a temperature of 37° C.

11.2—CHARACTERIZATION OF THE MICROPARTICLES:

The overall composition of the three batches of microparticles beforeredispersion ranges from: 5±1% polyamino acid; 39±3% Miglyol®; 55±3%PBS.

The particle size distribution of the microparticles, measured by laserscattering on a COULTER LS130 machine in FRAUNHOFER mode with “PIDS” ismonomodal for the three batches, with an average voluminal diameterD[4.3] of 2.4±0.5 μm and a standard deviation of 1.1±0.4 μm.

11.3—STUDY OF RELEASE:

To study the release, a known volume of microparticle dispersion isfiltered at a given time. The filtrate is then analyzed by HPLC to assaythe cytochrome released or not encapsulated. The filtrations werecarried out at T₀ and then after 3 hours, 5 hours, 17 hours, 24 hoursand 5 days of storage at 37° C.

FILTRATION CONDITIONS:

Stirred diafiltration cell of AMICON type, with a capacity of 50 ml.

Polysulfone type membrane (ref. XM 300, AMICON).

ASSAY CONDITIONS:

Affinity-separation HPLC on PLRPS column (300 A).

RESULTS:

The encapsulation yields, defined as the ratio of the amount ofcytochrome encapsulated to the amount of cytochrome initiallyintroduced, are 100%, for a cytochrome/polymer degree of encapsulationof 35%.

No release R of cytochrome into the dispersion medium is observed after5 days of storage at 37° C. (cf. FIG. 9).

Example 12 Microparticles Containing Cytochrome, Prepared from Miglyol®,Water and Poly(LEUCINE-CO-SODIUM GLUTAMATE) of 50/50 Composition and ofRandom Structure—Encapsulation by Self-assembly

12.1—METHODOLOGY:

A batch of microcapsules was manufactured according to the operatingconditions described in Example 4.1.1 and 4.1.2(i). The micro-particlesedimentate is then dispersed in a volume of 10 ml of phosphate-bufferedisotonic solution at pH 7.4 containing 1.0 mg/ml of horse heartcytochrome C. After a contact time of 24 h at ambient temperature, themicrocapsule dispersion is filtered according to the conditions given inExample 11. HPLC analysis reveals a free cytochrome concentration of 0.5mg/ml in the filtrate.

12.2—RESULT:

The degree of encapsulation of cytochrome is 10% relative to thepolymer, for an encapsulation yield of 50%.

Example 13 Microparticles Containing a Protein “OSP A” Which is anAntigen for Lyme's Disease, Prepared from Miglyol®, Water andPoly(EUCINE-CO-SODIUM GLUTAMATE) [sic] of 50/50 Composition and ofRandom Structure: Direct Encapsulation

13.1—METHODOLOGY:

13.1.1—STEP a):

PREPARATION OF THE GEL:

50 mg of poly(leucine-co-sodium glutamate) (abbreviated as LEU/GLU)lyophilizate synthesized according to Example 1 are introduced into ahemolysis tube.

450 mg of an aqueous solution containing 4.4 mg/ml of Osp A are added.

13.1.2—STEPS b) to e):

PREPARATION OF THE MICROCAPSULES:

These steps are carried out in the same way as that described for theprocess of Example 4.1.2(i).

FILTRATION:

A volume of 2 ml of microcapsule dispersion is introduced into anultrafiltration tube of ULTRAFREE XM300 type, sold by MILLIPORE.

After centrifugation, 1 ml of filtrate is collected and is analyzed byELISA assay.

13.2—RESULT:

The ELISA assay reveals an unencapsulated Osp A concentration of 0.21mg/ml in the filtrate.

The degree of encapsulation of Osp A is 0.36% relative to the polymer,for an encapsulation yield of 46%.

Example 14 Microparticles Containing a Glycoprotein “gD2t” Which is anAntigen for the Herpes Disease, Prepared from Miglyol®, Water andPoly(LEUCINE-CO-SODIUM GLUTAMATE) of 50/50 Composition and of RandomStructure—Direct Encapsulation

14.1—METHODOLOGY

14.1.1—STEP a):

PREPARATION OF THE GEL:

50 mg of poly(leucine-co-sodium glutamate) (abbreviated as LEU/GLU)lyophilizate according to Example 1 are introduced into a hemolysistube.

450±0.2 mg of an aqueous solution containing 0.666 mg/ml of gD2t areadded.

14.1.2—STEPS b) to e):

PREPARATION OF THE MICROCAPSULES:

These steps are carried out in the same way as that described for theprocess with emulsion inversion in Example 4.1.2(i).

FILTRATION:

A volume of 2 ml of microcapsule dispersion is introduced into anultra-filtration tube of ULTRAFREE XM300 type, sold by MILLIPORE.

After centrifugation, 1 ml of filtrate is collected and analyzed byELISA assay.

14.2—results:

the elisa assay reveals an unencapsulated gg2t [sic] concentration of 5μg/ml in the filtrate.

the degree of encapsulation of “gd2t” is 0.12% relative to the polymer,for an encapsulation yield of 92%.

What is claimed is:
 1. Microparticles for use as active principlevectors, comprising: a cohesive structure made of a physicochemicallystable and integral composite gel comprising oil, an aqueous phase, andat least one linear, noncrosslinked, synthetic copolyamino acidcomprising at least two different types of amino acid comonomers, ahydrophilic comonomer AA_(i) and a hydrophobic comonomer AA₀, saidmicroparticles having an average size which is controllable andadjustable over a range less than or equal to 500 μm.
 2. Microparticlesaccording to claim 1, wherein said cohesive structure is reflected by atleast one of the properties: a) absence of coalescence of themicroparticles after a lyophilization treatment, this non-coalescencebeing reflected by a conservation of the particle size distribution, inparticular of the D[4.3] in a proportion of ±20%, after rehydrationaccording to a test α₁; b) absence of coalescence on centrifugation,reflected by a conservation of the particle size distribution, and inparticular of the D[4.3] in a proportion of ±20%, according to a testα₂; c) resistance to pH variations, reflected by a conservation of theparticle size distribution of the microparticles and, in particular, ofthe D[4.3] in a proportion of ±20%, after exposure to pHs of 2 to 13,according to a test α₃; d) absence of coalescence in dispersion in abuffered saline solution, reflected by a conservation of particle sizedistribution of the microparticles, in particular of their D[4.3] in aproportion of ±20%, for storage times of greater than or equal to 3, 9and 18 months at temperatures of 37° C., ambient and 4° C. respectively,according to a test α₄.
 3. Microparticles according to claim 1, whereinthe copolyamino acid comprises: comonomers AA_(i) selected from thegroup of consisting of glutamic acid, aspartic acid, ornithine,arginine, lysine, asparagine, histidine and mixtures thereof, comonomersAA₀, selected from the group consisting of leucine, tyrosine,phenylalanine, valine, cystine, isoleucine and mixtures thereof. 4.Microparticles according to claim 3, wherein AA_(i)=Glu and/or Asp andAA₀=Leu and/or isoleucine, and/or Tyr and/or Phe.
 5. Microparticlesaccording to claim 1, wherein the copolyamino acid is of randomstructure or of diblock, triblock or multiblock structure. 6.Microparticles according to claim 1, wherein the copolyamino acid iseither multiblock and has a molar mass Mw of greater than or equal to5000 D, or random and has a molar mass Mw of greater than or equal to50,000 D.
 7. Microparticles according to claim 1, wherein the oil isformed of at least one fatty compound selected from the group consistingof a medium-chain fatty ester acid triglyceride of animal, plant orsynthetic origin, paraffin, polysiloxane oil, a fatty acid of animal orplant origin, and a fatty acid ester and/or salts thereof. 8.Microparticles according to claim 1, wherein the aqueous phase comprisesa saline solution.
 9. Microparticles according to claim 1, comprising:at least one medium-chain fatty ester acid triglyceride, deionized wateror buffered saline solution as the aqueous phase, the pH of the aqueousphase being between 6 and 8, copolyamino acid of Leu/Glu type. 10.Process for the preparation of microparticles according to claim 1,comprising the steps of: a) preparing a gel from the aqueous phase andat least one said copolyamino acid; b) placing the gel obtained fromstep a) in contact with at least one said oil to form a mixture; c)stirring the gel and oil mixture to form a dispersion of microparticlesin an oily or aqueous continuous phase; d) optionally separating themicroparticles and the oily or aqueous continuous phase; e) optionallyredispersing the separated microparticles in a storage liquid; and f)optionally lyophilizing the microparticles from step d) or redispersedmicroparticles of step e).
 11. Process according to claim 10, whereinstep a) comprises mixing the aqueous phase and the at least onecopolyamino acid in proportions such that the at least one copolyaminoacid represents from 2 to 50% by weight of the gel formed in step a).12. Process according to claim 10, wherein during step c), the oil isintroduced into the mixture from step b).
 13. Process according to claim10, wherein the stirring is carried out using a rotor/stator device andworking at a stirring speed of between 1000 and 40,000 rpm.
 14. Processaccording to claim 10, carried out at between 10 and 35° C.
 15. Processaccording to claim 10, wherein at least one active principle isincorporated into the oil and/or into the aqueous phase and/or into theat least one copolyamino acid and/or into the gel from step a), so as toobtain microparticles filled with the at least one active principle. 16.Microparticles according to claim 1, comprising at least one activeprinciple which is medicinal and is selected from the group consistingof proteins, peptides, polysaccharides and nucleic acids. 17.Microparticles according to claim 1, comprising an active principlewhich is at least one vaccine.
 18. Pharmaceutical specialty for oral,nasal, vaginal, ocular, subcutaneous, intravenous, intramuscular,intradermal, intraperitoneal, intracerebral or parenteraladministration, comprising microparticles according to claim
 1. 19.Microparticles according to claim 1, comprising at least one activeprinciple which is a plant protection or cosmetic product. 20.Microparticles according to claim 6, wherein the copolyamino acid ismultiblock and has a molar mass between 5000 and 100,000 D. 21.Microparticles according to claim 1, of a size between 0.05 and 100 μm.